VASCULAR SHUTDOWN AS AN EFFECT OF USING PHOTODYNAMIC
THERAPY TO TREAT CANCER
by
Elizabeth Mary Pascucci
A thesis submitted in partial fulfillment
Of the requirements for the degree
of
Masters of Science
in
Microbiology
MONTANA STATE UNIVERSITY
Bozeman, Montana
December 2008
©COPYRIGHT
by
Elizabeth Mary Pascucci
2008
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Elizabeth Mary Pascucci
This thesis has been read by each member of the thesis committee and has been
found to be satisfactory regarding content, English usage, format, citation, bibliographic
style, and consistency, and is ready for submission to the Division of Graduate Education.
Dr. Jean Starkey
Approved for the Department of Microbiology
Dr. Mike Franklin
Approved for the Division of Graduate Education
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a master’s
degree at Montana State University, I agree that the Library shall make it available to
borrowers under rules of the Library.
If I have indicated my intention to copyright this thesis by including a copyright
notice page, copying is allowable only for scholarly purposes, consistent with “fair use”
as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation
from or reproduction of this thesis in whole or in parts may by granted only by the
copyright beholder.
Elizabeth Mary Pascucci
December 2008
iv
TABLE OF CONTENTS
1. INTRODUCTION ..........................................................................................................1
Overview of Photodynamic Therapy ..............................................................................1
History of Photodynamic Therapy...........................................................................7
PDT and the Immune Response.............................................................................11
Cancer ...........................................................................................................................14
Two-Photon Photodynamic Therapy ............................................................................18
Previous Experiment Leading into This Project ....................................................21
2. MATERIALS AND METHODS..................................................................................22
In vivo Octreoate Cell Binding Experiments ................................................................22
Immunohistochemistry .................................................................................................25
Immunohistochemical Staining of Tumor Cells in Culture...................................26
Tumor Tissue Preparation......................................................................................29
Immunohistochemical Staining of Tissue Sections ...............................................30
Octreoate Binding/Uptake Experiment.........................................................................33
Octreoate Conjugation ...........................................................................................33
Cell Staining using Biotinylated Octreoate............................................................36
Tissue Staining using Biotinylated Octreoate........................................................38
3. RESULTS .....................................................................................................................41
Experiments using in vivo Octreoate Block..................................................................41
Immunohistochemistry .................................................................................................44
Immunohistochemical Staining of Tumor Cells in Culture...................................44
Immunohistochemical Staining of Tumor Tissue..................................................48
Cellular Binding/Uptake of Octreoate Experiments .....................................................51
Octreoate Conjugation ...........................................................................................52
Cell Staining using Biotinylated Octreoate............................................................54
Tissue Staining using Biotinylated Octreoate........................................................56
v
TABLE OF CONTENTS – CONTINUED
4. DISCUSSION ...............................................................................................................60
Limitations of This Study .......................................................................................66
5. CONCLUSIONS AND FUTURE DIRECTIONS........................................................68
REFERENCES CITED......................................................................................................70
vi
LIST OF TABLES
Table
Page
1. Spectrophotometer readings at the λ = 214
Absorption Reading Boundaries ...................................................................................53
vii
LIST OF FIGURES
Figure
Page
1. The Principle of Photodynamic Therapy ........................................................................2
2. The FDA Approved PDT Drug: Photofrin® ..................................................................4
3. PDT Induced Activation of Antigen Specific T Cells ..................................................13
4. RA 301 ..........................................................................................................................19
5. Amine-PEG 3 -Biotin......................................................................................................34
6. Images of Lung Tumors Taken In Vivo Following an Intravenous
Injection of High Molecular Weight FITC Dextran .....................................................42
7. Results of Evaluating Tumor Growth
Rates Post PDT Treatment............................................................................................43
8. T47D Cells Stained for the Somatostatin 2 Receptor ...................................................45
9. Staining results of T47D cells Fixed Before and
After Staining For the Somatostatin 2 Receptor ...........................................................46
10. Octreoate’s Effect on Somatostatin 2
Receptor Staining of T47D Cells................................................................................47
11. Results of NCI H69 Tissue Staining for the
Somatostatin 2 Receptor and vonWillabrand Factor ..................................................48
12. NCI H69 Tumor Tissue Double Stained Using Two
Primary Antibodies: Anti-SSTr2 and Anti-vWF ........................................................49
13. Results of A549 Tissue Staining for the
Somatostatin 2 Receptor and vonWillabrand Factor ..................................................50
14. A549 Tumor Tissue Double Stained Using Two
Primary Antibodies: Anti-SSTr2 and Anti-vWF ........................................................51
15. 96-well Immuno4 Plate Used to Confirm
Octreoate’s Successful Conjugation to Biotin ............................................................52
viii
LIST OF FIGURES - CONTINUED
Figure
Page
16. Tumor and Endothelial Cells Stained for Somatostatin
2 Receptors Using Biotinylated Octreoate..................................................................54
17. Bovine Endothelial Cells Grown in Tissue Culture Under
Experimental Growth Condititions and Stained for Somatostatin
2 Receptor Using Biotinylated Octreoate ...................................................................55
18. Lung Tumor Tissue Stained for Somatostatin
2 Receptors Using Biotinylated Octreoate..................................................................57
19. Lung Tumor Tissue Stained for Somatostatin 2 Receptors
Using Biotinylated Octreoate and Counterstained Using
Alexa Fluor 488 Phalliodin or Double Stained Using
Anti-vWF ....................................................................................................................58
ix
ABSTRACT
Photodynamic therapy (PDT) is a treatment that uses the combination of a
photosensitizing drug and light to selectively kill cancer cells. PDT has many potential
advantages such as minimal side effects, excellent cosmetic results, and no cellular
resistance burdening traditional cancer treatments such as chemotherapy and radiation.
Currently used in the clinic, a limitation is depth of light penetration; therefore, PDT can
only be used to treat superficial disease. Our novel PDT agent utilizes two-photon laser
technology, which increases the depth of light penetration, greatly increasing the
potential uses. Our PDT is able to kill selective cells because the PDT drug has a
targeting agent so the drug only goes to cancer cells that overexpress the Somatostatin
receptor 2 (SSTr2) on their cell surface. Laser light irradiates the cancer cells causing
cytotoxic singlet oxygen to be produced damaging the cells. It was observed, through in
vivo imaging of the tumors before and after treatment, that vascular flow was diminished
as a result of PDT. It is well established that angiogenesis must occur when a tumor
reaches a certain size in order for the cells to remain viable and the tumor to continue to
grow; therefore, vascular shutdown could be an important mechanism of how PDT
works. A series of experiments on the tumor vasculature using in vivo imaging,
immunohistochemistry and confocal microscopy has taken place since this observation to
determine what may be happening. Results of these studies have shown that tumor
vessels do express the SSTr2 and therefore effected by treatment. Experiments using a
somatostatin analog, octreoate, detected by a fluor has shown that the endothelial cells do
take the drug up. To ensure that it is blood vessels that were being studied, tumor tissue
was stained for both SSTr2 and vonWillebrand Factor (vWF), a recognized endothelial
cell marker. Staining patterns for both antibodies were similar. To strengthen the
argument, SSTr2- negative tumor cells also showed a positive staining pattern of their
vessels, demonstrating that even though the tumor cells are SSTr2- negative, the tumor
vessels can be SSTr2-positive and thus responsive to PDT.
1
CHAPTER 1
INTRODUCTION
Photodynamic Therapy
Photodynamic therapy (PDT) is an exciting approach to treating cancers and
various other diseases. PDT involves the use of a photosensitizing drug and light to
kill cells. A photosensitizer is a molecule that, when activated by light of the correct
and specific wavelength in the presence of oxygen, can generate reactive oxygen
species (9). PDT can be targeted to effect only cancer cells, producing death by
necrosis and/or via the programmed cell death pathway, apoptosis. The protocol
starts with an injected dose of the photosensitizing drug. If the drug is not designed
to specifically target the tumor cells, it still can preferentially accumulate within
tumor tissue versus normal tissue for a variety of reasons: elevated numbers of lowdensity lipoprotein receptors, presence of macrophages, decreased pH, abnormal
stromal tissue, leaky vasculature, high levels of newly synthesized collagen, and high
levels of lipids (2). Once the photosensitizing drug is taken up into the cells, a light
source is used to illuminate the area. This light source is most often a laser. The
drug, excited by absorption of the photons, undergoes one or more energy transitions
emerging in the triplet state. The triplet may participate in one-electron
oxidation/reduction reaction with a neighboring molecule which produces free radical
intermediates that react with oxygen (2). This is known as a type I photochemical
reaction. Alternatively or coincidentally, the more common type II photochemical
2
reaction can occur upon light absorption (Figure 1). In this reaction, the triplet-state
photosensitizer can transfer energy to ground state oxygen, generating highly reactive
and cytotoxic singlet oxygen (3). Due to the fact that singlet oxygen can migrate less
than 0.02 µm after its formation, PDT only acts locally to effect cancer cells. Singlet
oxygen exerts its irreversible cell damage by several methods: degrading lipids in cell
membranes; increasing prostaglandin E2 levels; generating haematin aggregates;
damaging the endothelial cells of blood vessels; inhibiting neoangiogenesis; and
directly releasing cytochrome C from mitochondria (4). The photosensitizer can be
regenerated for a subsequent activation. The success of PDT is multifactorial and
depends on a number of factors including efficacy of preferential uptake and retention
of the photosensitizer, correct wavelength and efficient absorption of light, (1) and
presence of oxygen in type II reactions.
Figure 1. The principle of photodynamic therapy. This figure describes the energy
transitions in the photosystem (PS) to ultimately create singlet oxygen (1O 2 ). Figure
used with permission from the author (5).
These reactions will only take place in cells where the photosensitizer has localized.
Therefore, this type of therapy presents a favorable treatment option to current cancer
treatments such as chemotherapy and radiation which damage cells throughout the
entire body. PDT is comparatively non-invasive, it can be done as an outpatient
3
procedure, repeated doses can be given without the worry of generating cancer cell
resistance, and the healing process results in little to no scarring (6).
Since the photosensitizing drug is such an important component of PDT, great care
and consideration is taken in designing and synthesizing these drugs. It is important
that the sensitizer be easy to administer systemically via injection. From this
consideration water soluble sensitizers might be expected to be best since blood is
water based (10). However, the sensitizer must also be able to enter cells, which
involves traversing the lipid membrane, so the drugs also need to have some
hydrophobic characteristics. Hydrophobic sensitizers are able to bind strongly to
lipoproteins in the blood stream and be transported selectively to malignant tissue
(10). Photosensitizers in use and under investigation are based on the tetrapyrrole
nucleus; examples include porphyrin (hematoporphyrin derivative (HPD) or
Photofrin® (Figure 2)), chlorins (benzoporphyrin derivative (BPD)), bacteriochlorins
(TOOKAD®), phthalocyanines (phthalocyanine 4 (Pc4)) and texaphyrins (Lutex®)
(10). In this study, the photosensitizer is a porphyrin compound (Figure 4).
Porphyrins are large molecules which make it possible to have one part of the
molecule be polar while another part is non-polar (7). Following endocytosis of
Photofrin® drug, the sensitizer molecules preferentially accumulate in the lipophilic
compartments of tumor cells such as the plasma membrane and endoplasmic
reticulum, but concentrations in the mitochondria are generally 1000-fold higher (8).
4
Figure 2. The FDA approved PDT drug: Photofrin® is a porphyrin molecule (10).
PDT kills tumor cells in a variety of ways. Directly, as described above, but
PDT also damages the vasculature if the photosensitizer is still in the vessels when
light treatment occurs. In addition, PDT activates the immune system through
localized inflammation. Studies of mRNA and protein expression before and after
PDT treatment have shown that PDT causes upregulation and downregulation of
many proteins. PDT-mediated oxidative stress induces an upregulation of early
response genes c-fos, c-jun. c-myc and egr-1 (2) inducing cellular responses
promoting apoptosis. Not surprisingly, based on the cellular insult of PDT, multiple
genes encoding stress-induced proteins are also activated post-PDT treatment. These
include heme oxygenase and heat shock proteins (11). In addition to these proteins,
PDT activates other factors important in immune function such as interleukin-6,
interleukin-10, and nuclear factor kappa B (2). Enhanced expression of early
response genes and stress proteins works to activate the immune system affecting
functions such as cell adhesion, antigen presentation and inflammation (2).
PDT is currently used to treat a variety of cancers and has the potential to treat
several others. PDT is the favored treatment of Barrett’s esophagus because of its
treatment benefits and economical factors. Although prognosis for lung cancer is
5
often poor, PDT can be used for this type of cancer, increasing survival and/or
improving quality of life for the patient. A Japanese clinical study using PDT on lung
cancer has shown promising results: complete response in 84% of patients and partial
response in 15% with tumor recurrence in only 11% of complete and partial response
patients combined (13). PDT can also be used in conjunction with other therapies
such as surgery. If a lung tumor is inoperable at presentation, PDT can be used to
shrink the tumor and convert it to an operable one (12). PDT has been used for
gastrointestinal cancers for over 20 years, debulking tumors as a palliative care
measure. PDT could be used on inoperable brain tumors giving patients a more
favorable prognosis. Based on mouse studies, PDT has a promising future in treating
malignant melanoma as well as non-melanoma skin cancer (12). Studies are
underway evaluating PDT’s success at treating eye diseases such as choroidal
melanoma and other intra-ocular tumors (12). Described here are just a few examples
of the uses of PDT. PDT has the potential to treat many types of cancers as a primary
treatment, in conjunction with other treatment methods, or as palliative care to
improve the quality of life for a cancer patient. Some interesting clinical trials are
underway to increase the success of bone marrow transplantation in acute leukemia
and non-Hodgkin’s lymphoma patients. Bone marrow transplants to these patients
often result in relapse because of tumor cell contamination. To help decrease the
instances of relapse, trials are testing PDT to eliminate tumor cell contamination of
the marrow. This method does not involve introducing toxic chemical agents or
6
foreign antibodies that would induce immune activation and prejudice the transplant
survival (12).
PDT has a promising future in cancer treatment. However, because of its
many benefits, it is also being studied as a treatment for other types of diseases.
Rheumatoid arthritis, psoriasis, and many eye diseases including age-related macular
degeneration (12) are a few examples of common and debilitating diseases that are or
could be treated with PDT. In addition, PDT can be used to treat infectious disease.
The number of antibiotic resistant strains of bacteria is increasing rapidly so the need
to find alternative treatment options is vital. Studies are underway to treat localized
infections of Methacillin-resistant Staphylococcus aureus and vancomycin-resistant
enterococci (10). The photosensitizers used to treat infections would be applied
topically and have different molecular frameworks to those used to treat cancers (10).
Photosensitizers used to treat infectious diseases include halogenated xanthenes (Rose
Bengal), phenothiazines (Toluidine Blue O and Methylene Blue), acridines and
perylenequinones (hypericin) (10). These photosensitizers have a low dark toxicity to
mammalian cells but when localized and activated are lethal to bacterial cells,
especially Gram (+) species (10). The same treatments options exist for other
microorganisms, viruses, fungi and yeasts (10).
There are numerous benefits of using PDT treatment; however, this treatment
method does have its limitations. A PDT set-up can be expensive. Some types of
lasers are expensive pieces of equipment that are very specific and do not really allow
the flexibility of using a variety of sensitizers since each is excited by a specific
7
wavelength. The light sources used clinically at this time do not operate in the near
infrared (NIR) tissue transparency window, so the treatment is limited to lesions that
are within 1 mm of the body surface. Two side effects that have limited the use of
PDT are nonspecific biodistribution and prolonged retention in tissue that forces
patients to avoid light for many weeks after treatment (9). Another limitation of PDT
is that it cannot be used to treat metastatic disease because it is not possible to
irradiate the entire body in a reasonable time frame (6). These restrictions can be
dealt with as further research reveals better targeted and faster clearing
photosensitizers and more effective timing of drug injection to light irradiation.
History of Photodynamic Therapy
In 1900 a German medical student named Oscar Raab discovered that light at
a certain wavelength in combination with the chemical acridine was lethal to
Paramecium caudatum (4). The first recorded case of using this method to treat a
cancer was three years later when H. von Tappeiner and A. Jesionek treated mice
with basal cell carcinoma with topically applied eosin and white light (5). One year
later, the method of using chemicals and light was coined “photodynamic action” (4).
The treatment was first tested on a human in 1912 when German scientist F. MeyerBetz injected himself with 200 mg of hematoporphyrin and experienced pain and
swelling of his skin after light exposure (4). In 1942, H. Auler and G. Banzar
discovered that porphyrins exhibited a modest preferential accumulation in tumor
tissue when tested on rats (4). The selection was only about 3 times that of normal
tissue (45). Throughout the years photosensitizers have evolved to become more
8
potent and better “targeted”. In 1960, R. L. Lipson and S. Schwartz observed that
injections of hematoporphyrin led to fluorescence of neoplastic lesions. These
scientists also treated hematoporphyrin with acetic acid to obtain a mixture called
“hematoporphyrin derivative” (HPD) that was better at localizing in the tumor tissue
and could be used for tumor detection (2). This HPD was purified and termed
Photofrin® (2).
Photofrin®, the first generation photosensitizer, has spearheaded the current
and future directions of PDT (Figure 2). The first country to approve Photofrin® was
Canada in 1993, for treatment of bladder cancer. Closely following this, were The
Netherlands and France, for treatment of advanced lung cancers (2). After clinical
trials in Japan from June 1989 to March 1992, the Japanese government approved
Photofrin® to treat early stage lung cancer in 1994. Japan was followed by Germany.
Many more countries in Europe quickly followed suit. Photofrin® was approved as
the first PDT drug by the United States FDA in 1995 to treat patients with high grade
dysplasia in Barrett’s esophagus, and then in 1998 for patients with non-small cell
lung cancer (2, 3). An advantage that Photofrin® had over the earlier
photosensitizers was that it was activated by light at 630 nm and thus could penetrate
moderately well into the tissue, especially compared to topically applied eosin (5). In
addition, Photofrin® accumulates moderately well in the mitochondria of tumor cells.
However, Photofrin® does not clear from the body quickly, and skin photosensitivity
persists for four to 12 weeks after treatment (5, 14). Photofrin® has been a great
starting place for clinical use of PDT; however, the ideal photosensitizer should be
9
chemically pure and of known specific composition (this is especially significant for
toxicity testing), have a high quantum yield for singlet oxygen production, have a
strong absorption at a long wavelength (for maximum tissue penetration), have
excellent photochemical reactivity, have minimal dark toxicity, and only be toxic in
the presence of the correct wavelength of light (4). The ideal photosensitizer should
also have strong preferential accumulation and retention in tumor tissue, clear rapidly
from the patient’s body after treatment, and be easy to synthesize and give to a patient
intraveneously (4).
In order to try to fulfill these requirements, a wave of second generation of
photosensitizers was created. Among these are Benzoporphryn Derivative (BPD)
which has a strong absorption at 692 nm and it is cleared from the body quite rapidly
through the feces. However, BPD is only somewhat selective in tumor accumulation
(14). Aminolevulinic acid HCl (ALA) (Levulan®) received approval in 1999 (5) and
is currently used to treat acne and other skin lesions. In the mitochondria, 5aminolevulinic acid is formed during the first step of the heme biosynthetic pathway
from glycine and succinyl-CoA (60). The last step of the pathway is the
incorporation of iron into protoporphyrin IX (PpIX), a compound that possesses
properties of an efficient photosensitizer (64). With the addition of exogenous ALA,
PpIX accumulates with some degree of selectivity and, with light application, PDT
can take advantage of this endogenous photosensitizer (64). ALA treatment differs
from the other photosensitizers discussed because it uses an endogenous
photosensitizer. Other PDT drugs discussed herein are considered exogenous
10
because the photosensitizer comes from a drug that is administered, not a substance
produced in cells. ALA’s biggest advantage is its rapid clearance of only 1-2 days,
but it is water soluble thus not able to enter cells well (5). Metatetra(hydroxyphenyl)chlorine (mTHPC) (temoporfin or Foscan®) is the most recent
photosensitizer to gain approval for use. It was approved in 2001 in Europe for the
palliative treatment of head and neck cancers. The absorption wavelength of mTHPC
is 652 and photosensitivity post-treatment only lasts 2-4 weeks (5). Another second
generation photosensitizer is known as WST09 or Tookad®. Tookad® is a
palladium-metalated bacteriopheophorbide that is activated at 763 nm (15).
Treatment with Tookad® targets the tumor vasculature making the “drug to light”
interval very short, so that the photosensitizer is still contained within the vessels
upon activation (15). Tookad® is only retained in tissue for three hours post
treatment (15). These second generation photosensitizers all have a longer absorption
wavelength for increased tissue penetration. Bis-amino (IV) phthalocyanine (BAMSiPc) is another second generation photosensitizer. It is a potent photosensitizer that
leads to tumor cell death through the apoptotic pathway (9). However, the
biodistribution and localization problems of BAM-SiPc may inhibit its clinical use
(9).
As second generation sensitizers are tackling the flaws of their prior
generation, third generation photosensitizers are coming to the forefront doing the
same for the second generation sensitizers. Third generation sensitizers are not only
improving tissue penetration and drug clearance, but also improving targeting to
11
increase selective affinity of the photosensitizer for the tumor tissue (9). Targeting
moieties, such as single-chain monoclonal antibodies, will enable the sensitizer to kill
tumor cells while minimizing healthy cell damage (9).
PDT and the Immune Response
Most of the common cancer therapies are immunosuppressive. The doses
required for chemotherapy and radiation to be effective are toxic to bone marrow, the
source of cells of the immune system (16). Major surgery can also have
immunosuppressive effects demonstrated by reduced cellular proliferation and
reduced secretion of interleukin-2 (IL-2) and gamma interferon (IFN-γ) by mitogenstimulated T lymphocytes (65). The decreases in cell proliferation and IL-2
production, but not IFN-γ production, appear to be the result of inhibitory factors,
such as prostaglandin E2 that are released from mononuclear phagocytes at elevated
levels after injury (65). In addition, alterations of monocyte functions have been
reported after major surgery, including loss of cell surface human leukocyte antigendr (HLA-DR) molecules as well as reduced secretion of cytokines, including IL-1, IL6, and IL-8 (65). The ideal cancer therapy would not only be able to kill the primary
tumor, but activate the immune system to recognize, find and destroy any residual or
metastatic tumor cells. PDT-induced anti-tumor immunity was first demonstrated by
Canti et. al. when he showed mice treated with PDT were resistant to subsequent
tumor challenges, and that cells isolated from tumor draining lymph nodes from PDT
treated mice could be transferred to naïve mice and carry with them this tumor
12
resistance (18). A separate study confirmed this finding, and found that anti-tumor
immunity resulting from PDT treatment was very active 40 days post-treatment (20).
The photooxidative lesions from a treatment session are recognized by the
immune system as altered self. Altered self activates the complement component of
the innate immune system. Complement is engaged by one of three pathways:
classical, alternative or lectin mediated, in response to pathogens, foreign cells, or
cells of the body that are altered and no longer recognized as self (17). Complement
opsonized cells that have undergone PDT treatment then attract neutrophils,
monocytes/macrophages, dendritic cells, and other immune cells displaying
complement receptors (17, 19) (Figure 3). Neutrophil infiltration at the tumor site is
essential for an immune response (21). Post-PDT treatment activated neutrophils can
remain within tumor vasculature causing endothelial cell damage which releases
cytokines and chemokines that will attract a fresh wave of immune cells (19, 17). In
addition, macrophages and dendritic cells are attracted to the tumor site and
phagocytosis of cancer cells occurs to generate antigen presenting cells. They present
tumor specific antigens to major histocompatibility class II molecules (19). The
presentation causes T lymphocytes to recognize and destroy tumor cells (19, 22); one
study found that this is the action of CD8(+) T cells, in particular (20). Since
neutrophils are so important in initiating the immune response, high levels of
neutrophils infiltrating the treatment site may serve as a positive clinical prognostic
indicator (21).
13
Figure 3. PDT induced activation of antigen specific T cells (16). Endothelial cell
damage and tumor cell death leads to inflammation, neutrophil infiltration,
chemokine and cytokine release and infiltration of immune cells. Figure used with
permission from the author and the Nature Publishing Group (NPG).
Immune activation from inflammatory and immune responses are key contributors to
the efficacy of PDT treating cancer (17). In fact, complete tumor resolution after
PDT treatment requires immune activation and function (19). PDT-induced immune
activation presents another aspect where PDT could be used as an adjuvant therapy,
possibly with a drug that also activates the complement system to enhance the
immune activation.
14
Cancer
In the year 2007, 1,444,920 people were diagnosed with cancer; another
1,437,180 people are expected to be diagnosed with cancer in 2008 (23). This year
565,650 Americans are expected to die of cancer, that is 1,500 people each day (23).
Cancer is the second most common cause of death in the US led only by heart
disease, and one in every four deaths was due to cancer (23). The overall cost of
cancer in 2007 was 219.2 billion dollars including costs for medical care and loss of
productivity (23).
The two types of cancer used in the current study were breast and lung. Of
the 1,444,920 people expected to be diagnosed with cancer this year, 184,450 cases
are expected to have breast cancer and 40,930 breast cancer patients are expected to
die (23). Breast cancer patients are usually women but occasionally can be men.
Risk factors for breast cancer include early menarche, never having carried a baby,
age, personal and family history (5% - 10% of people have a germline mutation of
BRCA 1 and BRCA 2 genes) (23). There are many treatment options depending on
the size of the tumor, type of cancer cells and stage of cancer progression. One
treatment choice is a lumpectomy which saves as much of the breast as possible, but
this treatment is limited to smaller tumors, and is often followed with radiation to kill
any cancer cells not removed by surgery. There are various types of mastectomy. A
partial or segmental mastectomy removes the tumor and some breast tissue along with
the fascia under the tumor. A segmental mastectomy saves much of the breast, but it
is often followed by radiation treatment (24). A simple mastectomy procedure
15
removes all the breast tissue, and is usually followed by treatments of radiation,
chemotherapy or hormone therapy (24). A radical mastectomy consists of removing
the breast, lymph nodes and chest muscles, but is rarely done now because the
procedure is so invasive and deforming. In addition, many problems follow a radical
mastectomy such as lymphodema and a high risk of infections. A modified radical
mastectomy is quite common and removes the entire breast, overlaying skin and
underarm lymph nodes (24). The first tumor draining lymph node is the sentinel
node, and a sentinel lymph node biopsy is common. If cancer cells are found in the
sentinel lymph nodes, these nodes are removed along with the axillary lymph nodes
to prevent further cancer spread (24). Breast reconstruction often follows
mastectomies. Other treatment options include radiation, chemotherapy, hormone
therapy and biological therapy. Radiation is used commonly after surgery to ensure
as much cancer cell kill as possible. It is also used for cancer that has invaded the
chest wall or that has spread to more than four lymph nodes in the axilla (24). Side
effects are unpleasant including lymphodema of the arm, damage to lungs, heart or
nerves, change in the breast tissue and an increased risk of developing another tumor
(24). Chemotherapy is used to decrease the chance that cancer will recur, to try and
control the spread of the disease, or to ease any symptoms the cancer mass is causing
(24). However, chemotherapy can cause hair loss, nausea, vomiting, fatigue, damage
to the heart, nerves, kidneys, and other organs, damage to immune cells,
“chemobrain” a condition that causes memory and concentration problems, premature
menopause and infertility (24). Hormone therapy works to decrease the chance of the
16
cancer returning and to reduce the tumor burden (24). There are two types of
hormone therapy. Selective estrogen receptor modulators (SERMS) block
endogenous estrogen from attaching to receptors on cancer cells, slowing tumor
growth and killing cancer cells. An example of this type of drug is Tamoxifen
(Nolvadex) (24). The second type of hormone modulators are the aromatase
inhibitors that stop estrogen production in cells other than the ovaries. Examples of
this type of drug include anastrozole (Arimidex), letrozole (Femara), and exemestane
(androstenedione) (24). These drugs increase the risk of osteoporosis and only postmenopausal women are eligible. Biological therapies take advantage of proteins such
as HER2-neu that are overexpressed on tumor cells or other proteins that are
overexpressed on tumor vasculature. These treatments use monoclonal antibodies to
block receptor sites and kill cells. Lapatimb (Tykerb) is approved to use in
conjunction with chemotherapy in women with advanced metastatic breast cancer
(24).
Of the 1,437,180 new cancer cases diagnosed in 2008, 215,020 cases are
expected to be lung cancer (23). Lung cancer usually forms from the cells lining air
passages. The two main types are small cell lung cancer and non-small cell lung
cancer and they are characterized by the size of the cell when viewed under the
microscope (23). Small cell lung cancers (SCLC) are less common, grow very
quickly, and have often already spread to other parts of the body by the time the
cancer is diagnosed (24). Non small cell lung cancer includes adenocarcinoma, the
most common type of lung cancer (30 – 40% of cases) and squamous cell carcinoma,
17
the second most common type of lung cancer (30% of cases) (24). It is estimated that
161,840 people will die from lung cancer this year (23). Lung cancer is the leading
cause of cancer deaths among Americans (24). Risk factors include smoking,
exposure to second hand smoke, radiation therapy of the breast or chest area,
occupational hazards such as asbestos, radon, arsenic, soot, tar, exposure to air
pollution (25). An additional risk factor emerging for lung cancer is genetics (66).
Studies have shown that two single nucleotide polymorphisms (SNP) on chromosome
15 increase the likelihood of developing lung cancer by 28-81% (66).
Treatment options include surgery, radiation, chemotherapy, and PDT (24).
The option a patient and his or her doctor select depends on if the type is small cell
versus non-small cell, tumor stage, and the patient’s general physical condition (23).
Surgery can range from minimally invasive to major. A thoracoscopy is a way to
access the chest through small incisions to diagnose and treat cancer (24). The other
surgery treatment options are more invasive because they require a major chest
incision to get to the lung tissue. These treatments range from a wedge resection
(removing a small section of the lung) to a lobectomy (removing an entire lobe of one
lung) to a pneumonectomy (removing an entire lung) (24). Radiation can be a
treatment option alone or with surgery. PDT using Photofrin® and a light delivered
using a bronchoscope is also a treatment option (24). Radiation, chemotherapy, and
PDT all have undesirable side effects as mentioned earlier.
18
Two-Photon Photodynamic Therapy
A major limitation of the current PDT treatments available is that the light
used is not able to penetrate any distance into the tissue, so only surface lesions and
lesions that can be reached using a scope are eligible for PDT treatment. This is
because the absorption of visible light in the tissue is high and this light scatters a
great deal in biological tissues (26). Two-photon (2PA) PDT was first proposed in
the 1930’s by Maria Goppert-Mayer, but difficulty developing a suitable
photosensitizer, and the requirement for expensive, large laser equipment, meant that
2PA PDT failed to attract a lot of attention until the 1960’s. Two-photon PDT is now
an emerging and promising specialty (26, 27). 2PA is a nonlinear optical process in
which two photons are absorbed simultaneously, and, by combining their energies,
promote the molecule to an excited state (26). 2PA porphyrin compounds can
generate singlet oxygen at much longer light wavelengths, about 800-1000 versus
400-700 of one photon compounds. Longer wavelengths are in the tissue
transparency window allowing for much better depth efficacy (26). In addition, 2PA
excitation requires a much higher power density than one photon so the laser beam is
more focused and consequently the laser can precisely illuminate only the area being
treated (26). In 1997 two studies were published demonstrating the near infrared
(NIR) excitation of a photosensitizer using a Ti: sapphire laser (26). These studies
used 9, 10-anthracenedipropionic acid (ADPA), an established singlet oxygen
scavenger in aqueous solutions. It reacts with singlet oxygen producing endoperoxide
19
and bleaching, and was used to show that 2PA did cause singlet oxygen production
(26).
Another limitation of the current PDT drugs is poor targeting. Second and
third generation sensitizers that use antibodies or large molecules to target the tumor
run a high risk of being taken up in the liver and causing harmful immunological
reactions (28, 29). Our novel 2PA PDT drug, RA 301, does not have those
limitations (Figure 4).
Porphyrin Photosensitizer
Targeting Peptide
NIR Imaging Component
Figure 4. RA 301. RA 301 is consists of three moieties: the center portion is the
porphyrin photosensitizer; the left is the NIR imaging component; and the right is the
targeting peptide.
RA 301 takes advantage of the fact that many tumors overexpress the somatostatin
receptor 2 (SSTr2) (Figure 4, component on the right). Somatostatin (SST) is a
regulatory peptide hormone produced and secreted by neuroendocrine and
inflammatory cells (30). It inhibits secretory and proliferative responses in target
20
cells by reducing the synthesis and secretion of local and systemic growth promoting
factors (31). The biological effect of SST is mediated through a family of five
specific high affinity G protein-coupled receptors (31). RA 301 is a 2PA PDT drug
comprised of three components: a small peptide targeting the SSTr2 (Figure 4, right
component), a NIR imaging agent to see in vivo where the drug is (Figure 4, left
component), and a porphyrin that is activated by light in the tissue transparency
window (Figure 4, center component) (28). Not only is SSTr2 overexpressed in a
large percentage of tumors, but the SST analog octreoate is easy to obtain
commercially. Octreoate is a SST analog that can mimic the biological actions of
SST. SST analogs are used to treat growth disorders such as acromegaly and treat
various endocrine tumors because they can bind to receptors as SST would and
inhibit the action of growth hormones (32).
RA301 is moderately soluble in the Solutol® excipient (BASF), and is given
to mice through tumor infiltration. In larger animals, the injection would be given
intravenously. This is because Solutol is very viscous and the dose needed to be
effective cannot be diluted enough for intravenous injection due to the small blood
volume of mice. Once in the body, the drug will accumulate only in the tumor cells
that are overexpressing somatostatin receptor 2. The deepest effective penetration of
our PDT protocol is not known because all animal work has been done using mice
that have a maximum body depth of about 2 cm. However, work in tissue phantoms
has shown cell kill at 4 cm deep (28). In experimental mice, RA 301 has been shown
to be cleared from the body after 24 hours (29). This drug is designed to solve
21
several of the problems of current PDT photosensitizers such as tumor cell targeting,
using longer light wavelengths for increased depth in the tissue transparency window,
and rapid clearance from the body after treatment.
Previous Experiment Leading into This Project
In vivo imaging is an important component of the projects done in the lab to
observe what is happening to the tumor. In our lab, select cancer cell lines were
transfected to express a luciferase gene. Luciferase is an enzyme used in the lab for
bioluminescence assays. When luciferase expressing cancer cells form a tumor it is
possible to visualize the cells using 0.02 ml of luciferin (1 mg/ 0.01 ml of PBS)
injected intravenously. The luciferin causes the enzyme luciferase to undergo an
oxidation reaction, producing light. It was observed through this type of in vivo
imaging, carried out before and after PDT treatment, that the tumor exhibited a large
amount of luciferase bioluminescence before PDT treatment and virtually none after
treatment. This observation led us to wonder whether we had killed all the cancer
cells and there were no cells left to express the bioluminescent signal. Or if there had
been a shutdown of the tumor vasculature as a result of PDT treatment and the
luciferin was not able to circulate to the tumor cells. The latter option was
investigated in this project.
22
CHAPTER 2
MATERIALS AND METHODS
In vivo Octreoate Cell Binding Experiments
This set of experiments elaborated on the experiment that led into this project.
They demonstrate the importance of our novel PDT drug binding to the somatostatin
receptors on the tumor cells for its effectiveness.
Mice used in this entire study were CB17 SCID. All animal procedures were
performed under general anaesthesia with inhaled isofluorane and were carried out
according to the NIH Principles of Laboratory Animal Care and approved by the
MSU Institutional Animal Care and Use Committee (IAUCAC).
The small cell lung cancer line, NCI H69, and the non-small cell lung cancer
line, A549, were propagated in tissue culture. The cell lines were grown in 10 ml
plastic cell culture flasks in DME/F-12 cell growth medium supplemented with 10%
fetal bovine serum, 0.12% sodium bicarbonate, 0.10 µg/L insulin, and cover
antibiotics. Cells were fed twice each week and when the cells reached a confluency
of approximately 80-90% they were subcultured. Subculture was carried out by first
removing spent cell medium and incubating the cells for 10 minutes at room
temperature in Tyrodes Ca2+Mg2+ -free saline, followed by a brief incubation at room
temperature in 0.05% Trypsin plus 0.02% EDTA. Adherent cells were rinsed off of
the flask with 1 ml of serum containing cell medium, also stopping further
trypsinization. One drop of new media plus cells was added to a new flask containing
23
10 ml of fresh medium. If, as in this case, the cells were needed for an experiment in
high numbers, the cells were subcultured into two to four 50 ml flasks. In this
experiment, both cell lines were needed to inject into mice to grow a tumor. As soon
as the cells reached approximately 80-90% confluency the flasks were subcultured as
described above, the cell suspension was added to a 50 ml plastic centrifuge tube and
was centrifuged for seven minutes at 1000 rpm. For injection subcutaneously in
mice, the cells were resuspended in complete medium at more than 107 cells per 0.1
ml/mouse.
The cell suspensions were kept on ice until injection. To grow tumors, 0.1 ml
of suspended cells plus 0.1 ml of BD Matrigel® Matrix (BD Biosciences) were
injected using a plastic 1.0 ml tuberculine syringe equipped with a 27 gauge needle.
Lung tumor cells were injected subcutaneously in the flank region.
When the tumors reached about 1cm3 or after adequate blood vessels had
formed for the tumor to be considered angiogenic the mice were anaesthetized and
treated with Nair® to remove hair on and around the tumor. The next day the tumors
were imaged to give a pretreatment baseline.
In vivo imaging was done by first injecting the mice intravenously via the tail
vein with 0.2 ml (10 mg per ml, 2 ml per kg body weight) 150,000 - molecular weight
(kilo Dalton) FITC dextran (34). This particular molecular weight dextran is large
enough that it will remain within the vasculature until it reaches the leaky vessels of
the tumor where it will extravasate within the tumor. An experiment to determine the
time post injection in which the highest concentration of FITC dextran accumulated at
24
the tumor site had been carried out previously. Following this earlier data, the mouse
was imaged at forty minutes post FITC dextran injection. Imaging was carried out
using a Kodak 2000MM scanner and equipped with a 449-500 nm bandpass
excitation filter and a 535 emission filter. Mice were scanned for 5 minutes. The
data was recorded using Kodak scanner software.
Since this experiment was designed to determine the importance of the drug
binding to somatostatin receptors on tumor cells for effective treatment, in one
experimental group of mice the somatostatin receptors were blocked using the
somatostain analog, octreoate (35). To block the receptors, 0.1 ml of octreoate (1 mg/
ml CMF) was injected intravenously just before PDT drug injection. The sensitizer
(100-200 µg) was infiltrated into the tumor in PDT treated mice. Photodynamic
therapy treatment was carried out on mice four hours after injection of the PDT drug.
A 2-photon NIR laser beam was rastered through the entire tumor in approximately
20-30 minutes, depending upon the size of the tumor (28). The average pulse energy
was measured at the tumor and was 847 mW at a 1 kHz pulse repetition rate. Pulse
duration was ca, 150-200 fs as measured using an auto-correlator (28). The laser
wavelength was 790 nm with a bandwidth of ca. 10 nm (28). During
experimentation, the laser beam was focused with a spherical lens of focal length f =
400 mm (28). The distance between the lens and the tumor was 200 mm so the
average density in the laser spot was ca. 5W/cm2 (28).
In vivo imaging after treatment was conducted in an identical manner to that
used to establish the pretreatment baseline in order to observe what effect PDT had on
25
the tumor vasculature. In addition to in vivo imaging to determine the results for this
experiment, the PDT effect on tumor volume was also measured and recorded.
Tumors were measured daily using a ruler to observe the effect treatment has on
tumor size. Tumor volume was calculated using the equation for the volume of an
ellipse. The equation is 4/3πabc where a is radii front to back, b is radii side to side
and c is radii top to bottom; all measurements were made in millimeters. There were
four groups of mice used in this experiment: mice treated with octreoate and then
PDT, mice treated with PDT, mice that were not treated in any way, and mice that
were treated with octreoate only. Each group had four replicate mice. Data analysis
was done after the experiment was completed by graphing the percent change in
tumor volume against time.
Immunohistochemistry
Fluorescent immunohistochemical staining was carried out on tumor cell lines
propagated in culture and tumor tissue from SCID mice to examine on a more in
depth level what was happening to the tumor and tumor vascular tissue as a result of
PDT treatment. Similar to the previous set of experiments, the somatostatin 2
receptor was the target of investigation since this is the receptor that the PDT drug
targets. All imaging was done on a Leica® TCS-NT using a 63X 0.9 NA oil
immersion objective, and either 488 nm or 568 nm laser excitation sources.
Fluorescence filter combinations used for fluorescein dyes (green) were TD
26
488/568/633, RSP 580, BP 525/50, and for rhodamines (red) were the same but using
a LP 590 instead of a bandpass emission filter.
Immunohistochemical
Staining of Tumor Cells in Culture
The breast cancer cell line, T47D, and the lung cancer cell lines, NCI H69 and
A549, were grown in culture as described in the previous section. The MCF7 breast
cancer cell line was also used in this study. Culture techniques for this cell line were
identical to that described for other cell lines, except that these cells required the
addition of 0.1% β-estradiol prepared at 10-5 M concentration to the medium. In this
experiment cells were grown on glass coverslips. To prepare them, 1.5 mm
coverslips were soaked in glacial acetic acid for 2 days. They were then taken out of
the acid and rinsed with water seven times followed by a wash using 7X detergent.
The coverslips were rinsed again using water seven times and then three times in DI
water. The coverslips were stored until use in 95% ethanol. The clean coverslips
were taken with tweezers and flamed to remove any remaining ethanol and placed in
a 10 mm Petri dish, one coverslip per Petri dish. The Petri dish was filled with 3 ml
of complete cell medium. When tumor cell cultures reached 80 – 90% confluency
they were harvested as previously described. One drop of the suspended cells was
added above the coverslip in each Petri dish. The cells were allowed to grow in a 7%
CO 2 in air gassed 37°C incubator.
When cells reached 70-80% confluency they were used for staining. All the
staining and rinsing steps took place in the Petri dish. To establish the best working
27
protocol for preserving and staining somatostatin receptors, we first determined
which the best fixation technique was. Three fixatives were tested on T47D and
MCF7 coverslip cultures: a) 100% ice cold acetone at 4°C for 10 minutes followed by
air drying for 20 minutes, b) 75% ethanol and 25% acetone for 20 minutes at room
temperature and c) 100% methanol for 20 minutes at room temperature. We also
examined post-fixation after the primary antibody and detection steps. It was
determined that acetone treatment before the antibody incubation was the best
fixative. However, 100% acetone was a little harsh on the cells, and, for the rest of
the experiments, we used acetone at a ratio of 75:25 in sterile DI water. Aldehyde
fixatives were not used to avoid the problem of autofluorescence.
Although there were modifications made to the procedure depending on the
specific experiment, the general method of staining cells on coverslips was as
follows:

Coverslips were rinsed two times with pre-warmed phenol red- free, serumfree DME/F12.

Cells were fixed in ice cold 75% acetone to 25% water for 10 minutes at 4°C
then air dried for 20 minutes.

Coverslips were rinsed twice in PBS buffer (36) and then once in PBS
containing 0.05% Tween 20 (36) (PBS-Tween).

Cells were incubated in the primary antibody for one hour at room
temperature. If a coverslip was not yet fixed it was incubated at 37°C in a 7%
CO 2 in air gassed incubator.
28

Coverslips were then rinsed three times with PBS-Tween.

Cells were incubated in the secondary detection antibody for 30 minutes at
room temperature. Again, if the coverslip was not yet fixed the cells were
incubated in a 37°C gassed incubator. The coverslips were kept in the dark
from this step on.

Coverslips were rinsed three times with PBS-Tween, twice in PBS and twice
in sterile DI water.

If the coverslip was not yet fixed at this point, it was fixed as described above
then rinsed twice in PBS-Tween, twice in PBS and twice in sterile DI water.

Coverslips were mounted on slides using Prolong Gold Antifade (Molecular
Probes).

Cells were examined using a Leica® TCS-NT laser scanning confocal
microscope equipped with a 63X oil immersion lens and the argon 488 laser
line was used for excitation. Images were collected using the Leica® confocal
software and MetaMorph®.
In this set of experiments, the primary antibody used was Rabbit Polyclonal antiSomatostatin 2 Receptor (SSTr2) (Advanced Targeting Systems) diluted 1:250 (final
working concentration was 4 µg/ml PBS) in 5% Normal Horse Serum; the detection
probe was Protein A-FITC (Pierce) diluted 1:50 (final working concentration was 20
µg/ml PBS) in PBS- 0.05% Tween. The anti-SSTr2 antibody receptor was developed
in a rabbit using a peptide corresponding to the extracellular domain of SSTr2
(Advanced Targeting Systems), therefore, membrane antibody binding is expected.
29
The Protein A-FITC works well as a detection agent because it binds to the Fc portion
of the primary antibody, and it is a bright fluor. A negative control omitted the
primary antibody.
The next experiment examined the ability of octreoate to block the antibody
staining for the SST2 receptor. Four experimental groups were used: group 1
coverslips were treated with 1 ml of 100 µg octreoate/ml serum free media for 1 hour
in a 37°C gassed incubator immediately after the fixation step, and were then stained
for the SSTr2 as previously indicated. Group 2 coverslips were incubated in 0.50 ml
of 100 µg/ml octreoate plus 0.50 ml of anti-SSTr2 primary antibody for 1 hour in a
37°C gassed incubator. Group 3 coverslips were incubated with the primary
antibody, then incubated in 1 ml of 100 µg/ml octreoate and finally incubated in the
detection agent. Group 4 coverslips were not incubated in octreoate at any time, but
were stained for the SST2 receptor as described above. Any coverslip not being
specifically treated during a particular step was incubated in PBS-Tween for that time
period.
Tumor Tissue Preparation
Tumor cell lines, T47D, NCI H69 and A549, were grown in culture as
described in the previous sections. In this experiment, the cells, once confluent in
their culture flasks, were subcultured, suspended, and injected subcutaneously into
mice as previously described. Lung cancer cells were injected subcutaneously in the
flank region and breast cancer cells were injected subcutaneously in the area of the
mammary fat pad. Tumors were allowed to grow until they were at least 1 cm3. The
30
mice were sacrificed and the tumor was dissected out. The tumor was trimmed to
only keep the areas that showed no signs of necrosis. The tissue was placed in a
cryomold (TissueTek®) and embedded in OCT compound (optimal cutting
temperature) matrix (TissueTek®) (37). The mold was placed in a petri dish and set
to float in liquid nitrogen until completely frozen. Frozen tissue was stored in a
-80°C freezer until needed.
When used for sectioning, the frozen molds were placed in the cryostat chamber
at -20°C along with the blade for one hour. Tissue was sectioned into 10 µm sections
(38) and picked up by melting onto a Superfrost Plus Gold slide. The slides
containing sections were stored in a -80°C freezer until needed.
Immunohistochemical
Staining of Tissue Sections
The most successful steps from staining cells on coverslips were modified to
create a protocol to stain tissue sections. Again, since somatostain 2 receptors are the
target of our PDT drug they were focus of this investigation. The general staining
procedure is as outlined below:

Slides were taken out of the freezer the day before staining and set out to dry
overnight at room temperature.

Sections were fixed in 75% ice cold acetone and 25% water for 10 minutes at
4°C and then air dried for 20 minutes.

Sections were washed three times in PBS and then twice in PBS-Tween.
31

If kidney sections were used in the experiment, endogenous biotin was
blocked at this point in the staining procedure. This was carried out as shown
below:
o 15 minutes at room temperature in 0.05% Biotin in PBS.
o 15 minutes at room temperature in 0.05% Streptavidin in PBS.
o Wash twice with PBS-Tween.

Sections were incubated in the primary antibody in a hydration chamber for
one hour at room temperature.

Sections were rinsed three times in PBS-Tween.

The primary antibody was detected by incubation in a fluorescent tagged
Protein A for one hour at room temperature. From this step onward the slides
were kept in the dark.

Sections were washed three times in PBS-Tween, twice in PBS and twice in
sterile DI water.

If the experiment called for double staining the second antibody was applied,
rinsed, and detected in the same manner as described elsewhere in this thesis.

Coverslips were mounted on top of the tissue sections using Prolong Gold
Antifade and left to dry.

Tissue sections were examined using a 63X oil immersion lens on a Leica®
laser scanning confocal microscope equipped with a 488 argon laser and a 568
krypton laser. The images were collected using Leica® confocal software and
32
MetaMorph® and adjusted to reduce background using Adobe® Photoshop
software.
A blocking step for nonspecific antibody binding was not necessary because the
tissue was taken from SCID mice that do not have endogenous antibodies. This
assumption was tested using a 5% Normal Horse Serum blocking step, and no
difference was seen in the staining results.
To begin, tissue sections were incubated with anti-somatostatin 2 receptor
primary antibody diluted 1:250 (final working concentration was 4 µg/ml PBS) in
PBS-Tween and detected using Protein A-FITC diluted 1:100 (final working
concentration was 10 µg/ml PBS) in PBS-Tween. When imaged, it appeared that
tumor vasculature was being stained. To be sure that it was vessels that were being
stained the next set of tissue sections were stained using rabbit polyclonal anti-human
vonWillebrand Factor (vWF) (Factor VIII) (Dako) diluted 1:100 (final working
concentration was 10 µg/ml PBS) in PBS-Tween for 1 hour at room temperature as
the primary antibody and detected using Protein A-FITC. vWF was used because it is
a well established endothelial cell marker (43). To further prove that vessels were
being stained by the anti-SSTr2 antibody, tissue sections were double stained for
somatostatin 2 receptor detected with Protein A-FITC and for vWF detected with
Protein A conjugated to Alexa Fluor 568 (Pierce). These staining procedures were
completed on SSTr2- positive tumor cells, NCI-H69 (39) and T47D (39), and on
SSTr2-negative cells, A549 (40). A negative control slide was used in each set of
experiments. This slide was not incubated in primary antibody.
33
Octreoate Binding/Uptake Experiment
This set of experiments was designed to examine if endothelial cells of the tumor
vasculature were taking up the PDT drug. These experiments could show if the
octreoate targeted PDT sensitizer was targeting tumor vessels because the endothelial
cells were expressing the somatostatin receptors.
Octreoate Conjugation
For this set of experiments octreoate needed to be conjugated to a material
that could bind to a fluorescent marker. To do this we decided to take advantage of
the extreme binding avidity avidin and biotin have for each other.
Avidin is an egg white glycoprotein. Avidin and its analogues streptavidin
and NeutrAvidin (Molecular Probes) have a very high affinity for biotin (K a is
approximately 1015 M-1) (41). NeutrAvidin is deglycosylated (41) and is
advantageous to use in staining procedures because there is less positive charge on
the surface of the molecule and the NeutrAvidin will not bind nonspecifically to
negatively charged cell surfaces and nucleic acids as much (42). Avidins are mainly
composed of β-pleated sheets and are tetramers of identical subunits each of which
contains a single biotin binding site (41).
The first step was to biotinylate octreoate. This was done using EZ-Link®
Amine-PEGn-Biotin (Pierce) (Figure 5). This is a water soluble biotinylation reagent
that features a polyethylene glycol (PEG) spacer arm and a terminal primary amine.
34
Figure 5. Amine-PEG 3 -Biotin (Thermo Scientific)
The procedure followed to do this was recommended by Pierce. Fresh 2-(Nmorpholino) ethanesulfonic acid (MES) buffer was made up by dissolving 70.4 g
MES free acid monohydrate and 193.3 g MES sodium salt in 800 ml of molecular
biology grade water. After mixing the volume was adjusted to 1000 ml and the pH
was adjusted to pH= 5.78. Five-2 mg aliquots of octreoate were dissolved in 1 ml of
MES buffer each. A 50 mM solution of Amine-PEG 3 -Biotin was prepared by
dissolving 21 mg in 1 ml MES buffer. Sixty µl of the Amine-PEG-Biotin solution
was added to the dissolved octreoate and mixed (this resulted in the addition of 3
µmol Amine-PEG-Biotin, a 100-fold excess over octreoate to ensure binding). A 100
mM solution of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC) (Thermo Scientific) was prepared in MES buffer by dissolving 19 mg in 1 ml
of buffer. This was completed quickly because EDC is very sensitive to moisture.
EDC works as a crosslinking agent to couple carboxyl groups to the –NH 2 group of
the amine-biotin, forming an amide bond. Three µl of the EDC solution was added to
the octreoate-Amine-PEG-Biotin solution and incubated for two hours at room
temperature with constant mixing. The tubes were centrifuged to remove any
35
precipitate that was formed during the reaction, unreacted biotinylation reagent and
any EDC by-products were removed by putting the mixture through a Bio-Gel PGDG desalting column.
To determine if the reaction was successful the liquid collections from the
desalting column were tested using an Immuno4 96-well plate and streptavidin
conjugated to Alexa Fluor 488 (Pierce). To do this 100 µl of each fraction was added
to one well of the 96-well plate. Non specific binding was inhibited using 180 µl of
5% Normal Horse Serum diluted in PBS incubated for 30 minutes at 37 °C. Wells
were rinsed four times using 200 µl of PBS. Wells were then incubated with 90 µl
streptavidin Alexa Fluor 488 diluted 1:100 (final working concentration was 10 µg/ml
PBS) in PBS for 30 minutes at 37 °C. The streptavidin Alexa Fluor 488 was
removed, and the wells were rinsed three times using 200 µl of PBS. Any remaining
liquid was shaken out onto a Technicloth wipe and the fluorescent levels of the plates
were checked on the Kodak Scanner equipped using excitation and emission filters
appropriate for FITC fluorescence. Presence of the biotinylated peptide was
confirmed by reading the fractions collected from the desalting column on a
spectrophotometer. The desired wavelength was λ= 214 to monitor the presence of
peptide bonds. Readings were taken of the MES coupling buffer, of blank tubes
containing only water and of each fraction from the desalting column. From these
readings the fractions were pooled into three samples. Sample 1, made up of the
material from the fractions with the highest λ= 214 spec readings, was kept. Samples
2 and 3 were made up of the next highest and the lowest λ= 214 readings respectively
36
and were frozen in a -80°C freezer. Sample 1 was dialysized to remove any residual
contaminants because the product was going to be used on live cells. This sample
was added to 1000 kD cut off pore size dialysis tubing (Spectrum Labs) and placed
into a 500 ml cylinder filled with a dilution of 1/30 PBS. Dialysis was conducted
with constant stirring. Three changes of 1/30 PBS were made at 20 minute
increments. The dialyzed product was frozen at an angle in a -80°C freezer and
lyophilyzed overnight. The lyophilyzed product was reconstituted in 2 ml sterile DI
water (10 ml biotinylated octreoate/ 2 ml water) and frozen in 250 µl aliquots.
Cell Staining
Using Biotinylated Octreoate
The breast and lung cancer cell lines, T47D and A549, were grown in tissue
culture on glass coverslips as described in previous sections. Once the cells reached
approximately 80-90% confluency they were ready to be stained. These cells were
stained using a similar procedure to that described previously for staining cells on
coverslips. In this experiment, however, the primary reagent was the biotinconjugated octreoate diluted 1:100 in PBS-Tween and the cells were incubated in a
37°C gassed incubator for 30 minutes. Detection was carried out using NeutrAvidin
Texas Red (Molecular Probes). The NeutrAvidin Texas Red was diluted 1:50 (final
working concentration was 20 µg/ml PBS) in PBS-Tween and incubated with the
cells for 30 minutes in a gassed incubator. In each set of coverslips, one was left to
be fixed after the primary reagent and detection incubations. The coverslips were
mounted on slides using Prolong Gold Antifade and left to dry before examination
37
using a confocal microscope equipped with a 568 krypton laser. Images were
collected using Leica® confocal software and MetaMorph® software.
Octreoate binding/uptake experiments were also done on endothelial cells
grown in culture. For this experiment, bovine aortic endothelial (BAE) cells were
used. The cells were grown in culture in an identical manner as the tumor cell lines
except that the medium consisted of Dulbecco’s Modified Eagles High Glucose
Medium (Sigma) plus 0.37% sodium bicarbonate, 10.0% fetal bovine serum, cover
antibiotics, 0.01 µg/100 ml insulin, 100 µg/ml heparin, and 250 µg/ml endothelial cell
growth supplement (Sigma). These cells were grown on glass coverslips, and stained
in the same manner as the T47D and A549 cells. A negative control in all
experiments omitted the octreoate incubation.
A second endothelial cell experiment was done to examine the effect of
growth factors on octreoate binding. Bovine aortic endothelial cells were grown in
culture on coverslips as described, except the medium in this experiment did not
contain endothelial cell growth factor supplement. Four treatments were used in this
experiment: a) one coverslip was grown in culture medium without added
supplements; b) one coverslip was grown in medium that was taken off of A549 cells
growing in the exponential phase in culture, filtered and mixed with an equal amount
of fresh culture medium. Another c) coverslip was grown in culture medium
supplemented with 10 ng/ml (46, 47, 48) murine vascular endothelial growth factor
(VEGF) (PeproTech Inc). The last d) coverslip was grown in cell medium
supplemented with 50 ng/ml (49, 50) recombinant human fibroblast growth factor
38
basic (bFGF) (R&D Systems). The cells were allowed to grow until 80-90%
confluent then were stained following the same procedure described previously. The
coverslip that was grown in unsupplemented medium was not only stained for SSTr2
using conjugated octreoate, but was also counterstained for polymerized actin using
Alexa Fluor 488 Phalloidin (Molecular Probes) diluted 1:250 (final working
concentration was 4 µg/ml PBS) in PBS-Tween for 30 minutes at room temperature.
After staining, the cells were examined using a Leica® laser scanning confocal
microscope equipped as previously described and images were collected using
Leica® confocal software and MetaMorph® software.
Tissue Staining
Using Biotinylated Octreoate
To further examine the octreoate uptake in tumor cells and tumor vasculature
the conjugated octreoate was incubated with tumor sections. NCI-H69 and A549
tumor cells were grown in culture and injected into SCID mice as previously
described. Once the tumors were of appropriate size, they were dissected out and
flash frozen as previously described. The frozen tumors were sectioned into 10 µm
sections using a cryostat at -20°C and picked up onto Superfrost Plus Gold slides. To
stain the tissue sections, the slides were taken out of the freezer the night before and
allowed to air dry at room temperature. The staining procedure was similar to that of
staining tissue sections as previously described except that, in place of the primary
antibody, biotinylated octreoate was used. Tissue sections were incubated in
biotinylated octreoate diluted 1:100 in PBS-Tween for one hour at room temperature
39
in a hydration chamber. Detection was carried out with an incubation in NeutrAvidin
Texas Red diluted 1:50 (final working concentration was 20 µg/ml PBS) in PBSTween, also for one hour at room temperature in a hydration chamber. Half the slides
were fixed before the application of the biotinylated octreoate and NeutrAvidin Texas
Red and half the slides were fixed after biotinylated octreoate and NeutrAvidin Texas
Red applications. The negative control slide omitted the incubation in biotinylated
octreoate.
The final two sets of experiments were designed to prove that tumor vessels
were binding octreoate. The first set of experiments was a double staining using
biotinylated octreoate detected with NeutrAvidin Texas Red followed by a
counterstain: Alexa Fluor 488 Phalloidin. This method allowed for better
visualization of the vessels and the cells because it stains the polymerized actin of the
cytoskeleton (44). The staining procedure was the same as the procedure already
described for double staining tissue sections, with minor changes. One group of
slides was fixed and incubated in biotinylated octreoate prepared as already
described. The slides were then stained with NeutrAvidin Texas Red prepared as
stated and counterstained using Alexa Fluor 488 phalloidin diluted 1:250 (final
working concentration was 4 µg/ml PBS) in PBS-Tween for one hour at room
temperature. A second group of slides was fixed, stained with Alexa Fluor 488
phalloidin, and then stained with biotinylated octreoate and NeutrAvidin Texas Red.
The third group was incubated in biotinylated octreoate and NeutrAvidin Texas Red
then counterstained and fixed last. The fourth group of slides was first counterstained
40
followed by the biotinylated octreoate and NeutrAvidin Texas Red staining and fixed
very last. The control slide was only incubated with NeutrAvidin Texas Red.
The second set of experiments were also done on NCI H69 and A549 tissue
sections and was also designed to ensure tumor vasculature was being labeled using
the biotinylated octreoate. These experiments utilized vWF as an endothelial cell
marker. The procedure was similar to one previously described for double staining
tissue sections. Five groups of slides were used. Group 1 was fixed, incubated in
biotinylated octreoate and NeutrAvidin Texas Red, and then incubated in anti-vWF
diluted 1:100 (final working concentration was 10 µg/ml PBS) in PBS-Tween for one
hour at room temperature in a hydration chamber. vWF was detected by another one
hour incubation in Protein A conjugated to an Alexa Fluor 568 diluted 1:50 (final
working concentration was 20 µg/ml PBS) in PBS-Tween. Slides in group 2 were
fixed, incubated with anti-vWF and Protein A-568 and then incubated with
biotinylated octreoate and NeutrAvidin Texas Red. Group 3 slides were first
incubated in biotinylated octreoate and NeutrAvidin Texas Red followed by anti-vWF
and Protein A-568 and fixed last. Slides in group 4 were stained with anti-vWF and
Protein A-568 first, then in with biotinylated octreoate and NeutrAvidin Texas Red
and, finally, fixed. The last group was fixed and stained with anti-vWF and Protein
A-568 only.
41
CHAPTER 3
RESULTS
Results from the studies described in the previous chapter are presented in the
same three section format. Each section focuses on a set of experiments highlighting
somatostatin 2 receptor’s role in PDT upon the cancer cells, the tumor vasculature
and PDT’s mode of action.
Experiments using an In Vivo Octreoate Block
These experiments used the somatostatin analog, octreoate, to block the
somatostatin 2 receptors on the cells to prevent the PDT drug from binding. The
evaluation of this experiment focused on the effect PDT had on the tumors when the
targeted receptors were blocked versus not blocked.
The first mode of evaluation was done using 150,000- molecular weight FITC
dextran to image the leaky tumor vasculature before and after PDT treatment. Mice
from two experimental groups were imaged (Figure 6).
42
A
B
C
D
Figure 6. Images of lung tumors taken in vivo following an intravenous injection of
high molecular weight FITC dextran. Images A and B were taken before and after
PDT treatment, respectively. Images C and D were taken before and after PDT
treatment, respectively; however, the mouse in this set of images received an injection
of octreoate prior to PDT drug administration.
The image sets in Figure 6 (A and B versus C and D) show a striking difference in
fluorescence intensity. The lack of fluorescence after PDT treatment in Figure 6b
compared to the image taken before PDT treatment (Figure 6a) indicates that the
FITC dextran was not able to circulate to the tumor indicates that the PDT is having
an effect on the tumor vasculature. The images in Figure 6c and Figure 6d
43
demonstrate a requirement for SST2 receptor binding in generating this vascular
effect. The fluorescence before and after treatment from the mouse that received an
injection of octreoate prior to the PDT sensitizer, is similar, indicating that, in this
case, PDT did not effect the tumor vasculature. These images demonstrate the
importance of the PDT drug’s ability to bind to the targeted receptors to cause
vasculature shutdown.
The second mode of evaluating the experimental octreoate block was to
follow the tumor growth rates. Tumor measurements started one day prior to PDT
treatment and continued until the tumors got too large for the mice to be maintained
on ethical grounds. The data was recorded as the percentage of growth as compared
to the initial volume against time. Figure 7 below presents the results from both
experimental replicates.
A
Days after PDT
NCI H69 Tumor Size (% of Initial Volume)
B250
225
200
175
150
125
100
75
50
25
0
B
Treated with Octreoate
and PDT
Mice Treated with PDT
0
5
10
15
20
Days after PDT
Figure 7. Results of evaluating tumor growth rates post PDT treatment. Panel A
depicts NCI H69 tumors of two groups of mice. The first group (blue) was not
treated. The second group (green) was administered the PDT drug and treated. Panel
B depicts NCI H69 tumors of two additional groups of mice. The first group (red)
was injected with octreoate followed by administration of the PDT drug and PDT
treatment. The second group (green) was administered the PDT drug and treated.
44
These graphs demonstrate the effectiveness of PDT. Mice treated with PDT
experienced tumor shrinkage followed by very little, slow tumor growth. However,
mice that received no treatment or a treatment of octreoate followed by PDT
exhibited rapid and extensive tumor growth. These data show that an octreoate block
in the tumors will decrease the PDT effect by being in direct competition for SSTr2
binding sites.
Immunohistochemistry
Immunohistochemical
Staining of Tumor Cells in Culture
Considering that staining for SST2 receptors was a new procedure in our
laboratory, an effective staining protocol needed to be developed. The first step was
to determine a fixation method that would preserve the SST2 receptors best. Three
staining methods were tested as mentioned in the previous chapter.
45
Figure 8. T47D cells grown on coverslips and stained for the somatostatin 2 receptor
using Rabbit Polyclonal anti-somatostatin receptor 2 followed by detection using
Protein A-FITC. Panel A depicts cells fixed using acetone at 4°C for 10 minutes and
air dried for 20 minutes at room temperature. Panel B depicts cells fixed using 75%
ethanol and 25% acetone for 20 minutes at room temperature. Panel C depicts cells
fixed using methanol for 20 minutes at room temperature. These are single scan
confocal images taken at the plane of best resolution.
These images in Figure 8 show the results of the different fixation trials. Acetone and
ethanol (Figure 8b) was not considered a good fixation technique because it did not
preserve the morphology of the cells well. Methanol (Figure 8c) seemed to preserve
the staining of the receptors on the cell surface, but not as well as acetone (Figure 8a).
In addition, cells fixed using acetone retained a more organized intracellular structure.
However, 100% acetone was considered too harsh because, when the ice cold 100%
acetone was added to the cells, many detached from the coverslip. Therefore, 75%
acetone was used as the fixative for our studies.
Another aspect of the staining technique that needed to be tested was the order
of fixation and staining. Fixation before and after anti-somatostatin 2 receptor
antibody staining was tested on T47D and MCF7 cells as shown below in Figure 9.
46
A
C
B
D
Figure 9. Staining results of T47D cells fixed before (panel A) and after (panel B)
antibody staining. Panel C and panel D are MCF7 cells fixed before and after
antibody staining and detection, respectively. These are single scan confocal images
taken at the plane of best resolution.
Punctate cell membrane staining was best observed using post fixation (Figure 9b and
Figure 9d). This observation conforms to expectations because somatostain is a
membrane-bound protein (30, 39) and the antibody used in this experiment is
designed to bind to the extracellular domain of the protein. Athough, punctate
membrane staining was observed on the cells that were fixed after the antibody and
Protein A-FITC application (Figure 9b and 9d), too many cells were lost from the
47
coverslips for this to be a routine procedure. From the results presented above for
both T47D and MCF7 cells it was determined that fixation before antibody staining
and detection was most useful.
Once the staining protocol was in place, we examined octreoate’s effect on
somatostatin staining at a cellular level in tissue culture. This experiment was
conducted using T47D cells grown on glass coverslips.
A
B
C
Figure 10. Octreoate’s effect on somatostatin 2 receptor staining of T47D cells. The
image in panel A is cells that were treated with octreoate before the anti-SSTr2
antibody; cells shown in panel B were treated with octreoate at the same time as the
anti-SSTr2 antibody, and the image in panel C is cells that were treated with octreoate
after the incubation with the anti-SSTr2 antibody. These are single scan confocal
images taken at the plane of best resolution.
These images show that octreoate has a very high affinity for the SST2 receptors,
actually replacing anti-somatostatin receptor 2 binding (Figure 10a) or out competing
the somatostatin antibody at the receptor site (Figure 10b and 10c). This is shown by
a much lower intensity staining compared to the images in Figure 9.
48
Immunohistochemical
Staining of Tumor Tissue
Having demonstrated positive SSTr2 staining in the tumor cell lines, the next
step was to stain for the SST2 receptors in tumor tissue. This experiment could help
to answer the question of whether our targeted PDT is affecting the tumor cells, the
tumor vasculature or both. NCI H69 is a cell line that is known to express SST2
membrane receptors and was tested first.
A
B
Figure 11. Results of NCI H69 tissue staining. Panel A depicts NCI H69 tumor
tissue that was flash frozen and sectioned into 10 µm sections and stained for
somatostatin 2 receptors. The anti-SSTr2 antibody was detected using Protein AFITC. Panel B depicts NCI H69 tumor tissue that was flash frozen and sectioned into
10 µm sections and stained for vWF. The anti-vWF antibody was detected using
Protein A-FITC. These images are each a reconstructed stack of confocal images.
The stained structures in the image shown in Figure 11, panel A resemble the pattern
of tumor vessels. It is also noteworthy to mention how much more prominently the
49
vessels are stained versus the NCI H69 cells. To confirm that vessels were being
stained, NCI H69 tumor tissue was also stained for vWF, a glycoprotein produced in
the endothelium and a well recognized endothelial cell marker. The staining pattern
in Figure 11, panel B is similar to that seen in Figure 11 panel A. To further convince
observers that tumor vasculature was being stained in both cases, NCI H69 tumor
tissue was double stained using both primary antibodies.
Figure 12. NCI H69 tumor tissue was flash frozen and sectioned into 10 µm sections
and stained for vWF (panel A) and somatostatin receptor 2 (panel B). The anti-SSTr2
antibody was detected using Protein A-568 and the anti-vWF antibody was detected
using Protein A-FITC. These images are reconstructed stacks of confocal images
taken simultaneously through separate laser channels.
The same staining patterns presented in both panels of Figure 12 confirm that the
tumor vasculature is being stained with anti-SSTr2 antibody.
The same staining procedures were conducted on a somatostatin receptor 2
negative cell line, A549. A549 tumor tissue was first stained for SSTr2.
50
A
B
Figure 13. Results of A549 tissue staining. Panel A shows A549 tumor tissue that
was flash frozen, sectioned into 10 µm sections and stained for SSTr2. The primary
antibody was detected using Protein A-FITC. Panel B shows A549 tumor tissue that
was flash frozen, sectioned into 10 µm sections and stained for vWF. The anti-vWF
antibody was detected using Protein A-FITC. These images are each a reconstructed
stack of confocal images.
Even though this cancer cell line is SSTr2 negative, the staining pattern is similar to
that expected for tumor vessels (Figure 13a). In addition, the pattern is similar to that
seen in Figure 11. Again, to confirm that vessels were being stained, A549 tumor
tissue was stained for vWF. The staining pattern in Figure 13, panel B visualizes the
tumor vessels. The vWF staining pattern in Figure 13, panel B is similar to the SSTr2
staining pattern of Figure 13, panel A. To further prove vessel staining for SSTr2,
A549 tumor tissue was double stained using both anti-SSTr2 and anti-vWF primary
antibodies.
51
A
B
Figure 14. A549 tumor tissue was flash frozen and sectioned into 10 µm sections and
stained for vWF (panel A) and somatostatin receptor 2 (panel B). The SSTr2
antibody was detected using Protein A-568 and the vWF antibody was detected using
Protein A-FITC. These images are reconstructed stack of confocal images taken
simultaneously using separate laser channels for each fluor.
From the results of staining A549 tumor tissue, the tumor vasculature of even SSTr2
negative cell lines can express somatostatin 2 receptors (Figure 13a and Figure 14b).
This is significant because it could allow SSTr2 negative tumors to be effectively
treated using our octreoate targeted PDT sensitizer. These results also provide insight
as to how our targeted PDT works, effecting not only cancer cells, but also the tumor
vasculature. Further experiments were done to confirm or reject this hypothesis.
Cellular Binding/Uptake of Octreoate Experiments
To answer the question of whether the targeted PDT photosensitizer was
getting taken up by the tumor vasculature we needed a “tagged” octreoate. Octreoate
was conjugated to biotin. In the primary reaction, octreoate conjugated to biotin
52
would bind to the somatostatin receptors. The detection method took advantage of
the high binding affinity that biotin and avidin have for each other. NeutrAvidin
conjugated to a Texas Red fluor was used in these experiments to visualize the
biotinylated octreoate.
Octreoate Conjugation
As stated previously, octreoate was conjugated to biotin using EZ-link AminePEG3-Biotin (Pierce) following the manufacture’s instructions. At the end of the
conjugation process, the sample was purified using a Bio-Gel P-GDG desalting
column. The fractions collected from the column were tested to demonstrate the
presence of the biotinylated product. To do this an Immuno4 96-well plate was used.
A small portion of each collection tube was added to one well. Detection utilized
streptavidin conjugated to an Alexa Fluor 488 (Invitrogen). The fluorescence of the
plate was checked on a Kodak scanner using filters appropriate for a 488 fluor.
Figure 15. 96-well Immunon4 plate used to confirm the success of octreoate’s
conjugation to biotin. Fluorescence of the samples in each of the wells was read
using a Kodak 2000 MM scanner equipped with a 448-500 nm bandpass excitation
filter and a 535 emission filter. The arrow indicates a well containing a positive result
for biotinylated octreoate.
53
If the well contained successfully biotinylated material the sample would bind to the
well and the fluorescent streptavidin would bind sample in that well. The bound
streptavidin would be detected by the scanner. Figure 15 shows an example positive
well (bright well with the arrow) and several negative wells (wells that are dark and
non-fluorescent) from the scan. Light scattering was evident around the edges of the
negative wells, a normal effect from the scanner.
Presence of the biotinylated peptide was confirmed by reading the samples
collected from the desalting column on a spectrophotometer at the λ = 214 absorption
for peptide bonds. Based on the λ = 214 reading the fractions eluded from the
desalting column were divided into three pooled samples (Table 1).
Table 1. λ = 214 reading boundaries. The sample’s reading must fall within these
boundaries to qualify for a sample designation. These readings were compared with
control readings: MES coupling buffer has a λ = 214 of 1.17 and water has a λ = 214
reading of 0.01.
Sample
Low Spec Reading
High Spec Reading
Sample 1
1.47
1.48
Sample 2
1.02
1.46
Sample 3
0.08
0.66
Sample 1 had the highest λ = 214 readings compared to the positive control λ = 214
reading of MES coupling buffer so these collection samples were pooled as a positive
conjugation result. Sample 1 (positive for biotin in the plate assay and showing
peptide bond absorption at λ = 214) underwent further purification, was lyophilized
54
and reconstituted in 2.0 ml of water (a final concentration of 5 mg/ ml) for
experimental use.
Cell Staining
Using Biotinylated Octreoate
Experiments, to see if octreoate was taken up by tumor cells and the cells of
the tumor vasculature, were initiated on cells grown on glass coverslips in tissue
culture. The cells were incubated with the biotinylated octreoate and the detection
utilized NeutrAvidin- Texas Red.
A
B
C
Figure 16. Tumor cells grown on coverslips and stained for somatostatin 2 repectors
using biotinylated octreoate. The biotinylated octreoate is detected using
NeutrAvidin-Texas Red. The cell lines are as follows: panel A shows T47D cells,
panel B is A549 cells and panel C is bovine aortic endothelial (BAE) cells. Each of
these images is a reconstructed stack of confocal images.
The structural similarities between the cells are apparent by observation. Each cell
line exhibits strong peri-nuclear punctate staining. Overall staining was most intense
for the SSTr2- positive T47D cell line (Figure 16a). In particular, T47D cells
exhibited a more defined cellular membrane staining than either A549 (Figure 16b) or
BAE (Figure 16c) cells.
55
Since tumor vascular endothelium exists in a milieu containing proangiogenic
factors, the next experiment examined how endothelial cell growth factors might
affect the SSTr2 staining of BAE cells. BAE cells were grown on glass coverslips
and subjected to a variety of different growth conditions. The growth factors used
were 10 ng/ml bFGF, 40 ng/ml VEGF, medium conditioned by proliferating A549
tumor cells and complete endothelial cell medium absent of any added growth
factors. Somatostatin 2 receptors were stained using biotinylated octreoate and
detected using NeutrAvidin- Texas Red.
A
C
B
D
E
Figure 17. BAE cells grown on glass coverslips in medium containing different
growth factors. Panel A shows cells in medium not containing any added growth
factors and panel B shows the same cells as in panel A with an Alexa Fluor 488
Phalloidin counterstain. Panel C shows cells grown with added bFGF in the medium;
panel D shows cells grown with added VEGF in the medium; and panel E shows cells
grown with tumor conditioned medium. White arrows in this figure point out areas of
high intensity staining indicating increased SSTr2 production. Each of these images
is a reconstructed stack of confocal images. Images in panels A and B were taken
simultaneously using separate laser channels for each fluor.
56
The bFGF and VEGF did affect the endothelial cells. Not only did the cells grow
more rapidly in culture, exhibited by an increased number of mitotic figures and a
shorter time of growth to reach confluency, but the cells exhibited more perinuclear
staining (white arrows in Figure 17c and Figure 17d). The endothelial cells also grew
more rapidly in tumor conditioned medium, although this effect was less than for the
isolated growth factors. These cells also showed more perinuclear staining (Figure
17e). The cytoskeletal counterstain was done to examine if octreoate was localizing
with actin components of the cell cytoskeleton (Figure 17b). In all conditions, there
was weak cell membrane staining.
Tissue Staining
Using Biotinylated Octreoate
To observe octreoate binding/uptake into endothelial cells of the tumor
vasculature, tumor tissue sections were stained using the biotinylated octreoate. NCI
H69 and A549 lung tumor tissue was used in this experiment.
57
A
B
Figure 18. Lung tumor tissue was cut into 10 µm sections and stained for
somatostatin 2 receptors using biotinylated octreoate detected with NeutrAvidinTexas Red. Panel A is a NCI H69 tissue section. Panel B is an A549 tissue section.
Images in each individual panel are a reconstructed stack of confocal images.
The staining patterns depicted in both panels of Figure 18 mimic the staining patterns
of previous tissue sections of confirmed tumor vessels. The staining pattern observed
in both panels of Figure 18 also imitates the pattern expected of vessels. The tumor
vasculature staining was confirmed by double staining lung tumor sections in two
separate procedures. The first stained for somatostatin receptors using biotinylated
octreoate and an Alexa Fluor 488 Phalloidin counterstain (Figure 19a and 19b), and
the second stained for somatostatin 2 receptors using biotinylated octreoate and a
primary ant-vWF antibody (Figure 19c and 19d).
58
C
A
D
B
Figure 19. Panel A and panel B depict A549 tissue samples cut into 10 µm sections
and stained for SSTr2 using biotinylated octreoate detected using NeutrAvidin- Texas
Red (panel B) followed by a counterstain using Alexa Fluor 488 Phalloidin (panel A)
to detect polymerized actin. The inset panel C and panel D depict a putative vessel in
a NCI H69 tissue section double stained using biotinylated octreoate detected using
NeutrAvidin- Texas Red to stain for SSTr2 (panel D) and for vWF using anti-vWF
primary antibody detected using Protein A-FITC (panel C). Images in each
individual panel are a reconstructed stack of confocal images. Images of panels A
and B as well as panels C and D were taken simultaneously through separate laser
channels for each fluor.
59
The counterstain using Phalloidin Alexa Fluor 488 (Figure 19a) shows how little
polymerized actin the tumor cells have, while the endothelial cells of the proposed
tumor vasculature exhibit a much more prominent staining pattern. The actin staining
pattern of the counterstain matches the staining pattern of the conjugated octreoate
staining for SSTr2 (Figure 19b). The double staining images of NCI H69 tumor
tissue is not shown but results were as expected in that the small round cells do not
have a lot of polymerized actin, and therefore there was weak counterstain signal.
The inset images depict NCI H69 tumor tissue double stained for vWF (Figure 19c)
and for SSTr2 using biotinylated octreoate (Figure 19d). The membrane staining
SSTr2 pattern matches the vWF staining pattern, and these images show that the
biotinylated octreoate is binding to receptors on endothelial cells of this tumor vessel.
60
CHAPTER 4
DISCUSSION
Photodynamic therapy is a cancer treatment which uses the combination of a
photosensitizing drug and light to selectively kill cancer cells. PDT promises to be
important in the future of cancer treatment in part because the PDT drug can
selectively accumulate in the tumor microenvironment, or can be made to target the
cancer cells. Thus, the treatment only affects the cancer and not the entire body,
unlike many other cancer treatment options that are available. Studies have shown
that PDT not only acts on cancer cells by producing cytotoxic singlet oxygen, but also
activates the immune system, and can destroy tumor vasculature depriving the tumor
cells of essential oxygen and nutrients.
Angiogenesis is a physiological process involving the growth of new blood
vessels from pre-existing ones (51). Angiogenesis is a normal, but highly regulated,
process in growth and development occurring most frequently during wound healing,
embryonic development, and in the female reproductive cycle (53). The role of
angiogenesis in cancer development is well accepted. If a tumor mass is to grow
beyond a few millimeters in size the “angiogenic switch” must occur (53).
Angiogenesis is initiated from the production and release of angiogenic growth
factors from cancer cells (52). Examples of angiogenic growth factors are VEGF and
bFGF which are used in this study. Upon stimulation from these growth factors
endothelial cells become activated. The activated endothelial cells break down the
61
underlying basement membrane, proliferate and migrate. The migrating cells attach
to integrin adhesion molecules and sprout new blood vessels (60).
Tumor vasculature represents an important target for cancer therapy and
second generation photosensitizers are taking advantage of the tumor cell dependence
on a functional blood supply for continued growth (59). Photosensitizers such as
Tookad® are being used to treat cancer by damaging the tumor vasculature. In the
clinic, the drug is injected intravenously and, immediately following this, the tumor
area is illuminated. The drug is still contained within the vessels and the generation
of singlet oxygen causes endothelial cell killing (15). The destroyed tumor
vasculature can no longer supply the tumor with oxygen and nutrients and the tumor
cells die of starvation.
Several tumor cell lines used in the lab are engineered to express the
luciferase gene. As the cells grow in mice, tumors can be monitored by injecting
luciferin and visualizing the resultant bioluminescence using in vivo imaging. The
luciferase activates a chemical reaction of the luciferin producing light. Tumors were
monitored in this manner before and after PDT treatment and it was observed that
there was more light produced before treatment than after. This observation led to
two possible conclusions: a) cancer cells were destroyed or b) the luciferin was not
circulating to the tumor due to damaged tumor vasculature. The experiments carried
out during the current study focused on determining if our novel PDT sensitizer could
combine the benefits of not only targeting the cancer cells but also destroying the
tumor vasculature.
62
The first set of experiments utilized in vivo imaging, after intravenously
injected FITC dextran, to examine the integrity of the tumor vasculature before and
after PDT treatment. Octreoate was used to block somatostatin 2 receptors and
prevent our PDT drug from binding. Without the octreoate block, the images show a
remarkable difference in the amount of fluorescence before treatment (Figure 6a) and
after treatment (Figure 6b) image. In contrast, when octreoate was used to block
SSTr2 the amount of fluorescence detected was similar (Figure 6c and d). Without
the octreoate block the lack of fluorescence after treatment indicated that the FITC
dextran was not able to circulate to the tumor because the vasculature had been
damaged. The effectiveness of PDT treatment was confirmed by measuring and
evaluating tumor growth following treatment in mice. As observed in Figure 7, mice
that received no treatment or an octreoate injection followed by PDT treatment
experienced rapid and extensive growth of the tumor. Mice that received PDT
treatment exhibited initial reduction in size of the tumor followed by much slower
tumor growth. These experiments show the efficacy of the targeting moiety of the
PDT drug. When unable to bind to somatostatin 2 receptors the drug is ineffective,
and treatment using PDT is not successful. Similar amounts of FITC dextran delivery
in the tumor before and after treatment in the octreoate treated mice indicated that the
vasculature was not damaged during treatment; furthermore, tumor growth rates
demonstrated that PDT treatment was not effective after octreoate block.
The next set of experiments focused on using tumor cells in culture. SSTr2positive and SSTr2- negative cell lines were stained using an anti-SSTr2 antibody to
63
demonstrate the presence of receptors on the tumor cells. SSTr2- positive breast
cancer cell line, T47D, exhibited punctate membrane staining in addition to
prominent nucleolar staining. Punctate membrane staining can be seen when staining
for many tumor cell receptors because receptors are not uniformly distributed on the
surface. Staining procedures may also induce aggregation of receptors. Overall
staining was much weaker with the introduction of octreoate, demonstrating the cell
line does express the SST2 receptors on its cell surface and, therefore, can be targeted
by our PDT drug.
After observing the positive tumor cell staining, the next step was to examine
tumor tissue. By studying tumor tissue, both tumor cells and tumor vasculature could
be examined. Images of tumor tissue showed positive SSTr2 staining patterns of the
tumor vasculature in both SSTr2- positive NCI H69 tumor sections (Figure 11a) and
SSTr2- negative A549 tumor sections (Figure 13a). The vascular staining of the NCI
H69 tissue section was much more prominent than the staining of the tumor cells. It
has been reported that SSTr2 expression is upregulated several-fold in vasculature
located within 2 cm of a tumor (56). The positive SSTr2 staining of the A549 tumor
vasculature could expand the use of our targeted PDT sensitizer because treatment
would not be limited to tumors whose cells overexpress SSTr2. If the endothelial
cells comprising the tumor vasculature are SSTr2 positive and can bind the drug they
will be susceptible to PDT killing. The strong SSTr2 staining of the tumor
vasculature could indicate an important mechanism through which our PDT protocol
treats cancer.
64
In the double staining experiments of tumor tissue, it would have been
beneficial to have an additional negative control in which each primary antibody was
used individually with the sequential application of both the Protein A-FTIC and the
Protein A-568. This would examine the possibility that in the double staining
experiments, using two primary antibodies, each detected using a Protein A, that
Protein A-FITC and Protein A-568 were detecting the same antibody.
Other studies have determined that angiogenic blood vessels of tumors do
express SSTr2 at the gene level but it was initially unclear if the genes were translated
into proteins (54). More recently, it was reported that endothelial cells in
proliferating human blood vessels express SSTr2 (55, 58). Since angiogenesis is
occurring during tumor development, the tumor vessels will express SSTr2 and will
be targeted by our PDT agent. Normal endothelial cells would not be affected by
SSTr2 targeted PDT because they are normally quiescent, dividing only once every
2–5 years (61).
The next question was if our targeted PDT drug was getting internalized into
the endothelial cells. For this experiment the somatostatin analog, octreoate, was
conjugated to biotin. The biotin’s only function was in detection; the detection
method in these experiments used NeutrAvidin conjugated to a Texas Red fluor. Cell
staining patterns from this experiment show organized punctuate membrane staining
from T47D cells (Figure 16a). However, the staining patterns of the endothelial cells
used in this experiment more closely resemble the intricate peri-nuclear staining
shown in the A549 cells (Figure 16c and b). This could be due to the fact that
65
proliferation in cell culture may not be accompanied by surface expression of SSTr2.
Based on the literature (40), A549 cells were used as the SSTr2- negative cell line our
experiments. However, the staining pattern obtained in the results of this experiment
would seem to indicate that A549 cells are producing the somatostatin 2 receptor.
These contradicting observations are resolved with the observation of definite perinuclear staining but no membrane staining concluding that the A549 tumor cell line
does produce SSTr2 but does not express these receptors on the cell surface.
The next experiment was designed to test aspects of the tumor
microenvironment to examine if membrane SSTr2 staining could be induced in BAE
cells. Endothelial cell growth factors were added to the media and the cells were
grown in culture and stained the using biotinylated octreoate and NeutrAvidin Texas
Red. In culture, VEGF and bFGF as well as A549 conditioned media caused the
BAE cells to grow approximately twice as fast. The images obtained from this
experiment showed exaggerated peri-nuclear staining (Figure 17) when compared to
cells grown in normal medium conditions. Increased peri-nuclear staining signifies
increased production of SSTr2. Lack of clear membrane staining, however, indicates
that the growth factors used do not stimulate membrane localization of endothelial
SSTr2 production. Although VEGF and bFGF were obvious choices to begin
experimentation (33), others of the over 20 documented endothelial cell growth
factors likely lead to SSTr2 membrane localization. If the study were repeated,
appropriate choices might be transforming growth factor- alpha (TGF-alpha),
pleiotrophin (PTN) or interleukin-8 (IL-8). Conditioned medium from the A549 cell
66
line was chosen because these cells do not express somatostatin 2 receptors on their
cell surface simplifying analysis of results. If conditioned medium from a different
tumor cell line was used it could easily lead to different results. Should the study be
repeated, conditioned medium would also be taken from a cell line that does express
SSTr2 on its membrane, such as T47D.
Images obtained from staining for somatostatin 2 receptors, using the
conjugated octreoate in NCI H69 and A549 (Figure 18) tumor tissue sections, show
positive SSTr2 staining. The staining pattern mimics that of tumor vasculature
indicating the octreoate was bound by the endothelial cells. The vessels exhibit
stronger staining patterns than the tumor cells. A study completed by Watson et.al.
using a radiolabelled somatostatin analog also showed vascular uptake and indicated
that this would enable tumor imaging of SSTr2- negative tumors (55). This provides
another use for targeting the SSTr2 on tumor vasculature.
Limitations of This Study
In the course of this study we found a limitation involving confocal
microscope equipment. In the experiment involving using a dye to detect octreoate
and its uptake within the cancer cells, the dye desired for use was the Licor CW 800
because it is the NIR imaging fluor that is going to be used in future whole animal
studies. However, this dye could not be used because, although MSU has a confocal
microscope that could excite the Licor CW 800 dye, MSU does not have a confocal
microscope with detectors sensitive enough in the NIR region of the spectrum to
67
detect the emission of this dye. Therefore, Texas Red was used as the fluor in the
uptake experiments, instead.
68
CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS
Vascular targeted PDT sensitizers represent one of the newest generation of
photosensitizers (62). However, the preference of vascular versus cellular targeting is
highly dependent upon the location of the photosensitizer at the time of light
illumination (63). In most cases treatment relies on strategic drug – light interval
timing and not on strategic targeting using drug moieties. Based on experimental data
it is evident the endothelial cells comprising the tumor vasculature express SSTr2.
Our novel PDT drug is unique in not only targeting tumor cells but targeting the
tumor vasculature as well. Targeting, treating and shutting down the tumor
vasculature are important mechanisms in the way our PDT protocol treats cancer.
PDT has the potential to decrease morbidity effects from treatment and
improve quality of life for patients with many health conditions and diseases. It also
has the potential to improve health economics (6). Future improvements involve
using the immune system activating properties of PDT in combination with other
immunotherapy protocols to control tumor growth on a more long term basis (2). As
new, better, photosensitizers are developed, pharmaceutical companies will look to
expand the conditions and diseases PDT can treat. This involves improved drug
delivery systems for better tumor targeting, such as, using liposomes, ligand-based
targeting with, for example, insuln, and using growth factors or adenoviruses (4).
69
PDT could also be improved by using new light sources to increase penetration into
the tissue.
Future directions for our PDT study will involve larger animal (canine and
rabbit) testing to accurately measure the maximal depth of tissue penetration that is
possible using 2PA PDT. Increased depth of penetration will expand the potential
uses for 2PA PDT because it would allow treatment of tumors that are located deep
within the body and/or are not accessible by minimally invasive surgery.
70
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